Humanized antibodies specific to the protofibrillar form of the beta-amyloid peptide

ABSTRACT

The present application relates to humanized antibodies specific to the protofibrillar form of the beta-amyloid peptide, and to the use of said antibodies in the field of Alzheimer&#39;s disease.

The present invention relates to humanized antibodies specific for theprotofibrillar form of β-amyloid peptide. The present invention alsorelates to the therapeutic, diagnostic and/or preventive use of theseantibodies, in particular associated with the induction and with theprogression of neurodegenerative disorders and/or with diseasesassociated with the deposition of amyloid plaques, and notablyAlzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease thataffects a high proportion of the older population. This disease ischaracterized clinically by memory loss and a decline in cognitivefunctions, and neuropathologically by the presence, in the brain, ofintracellular neurofibrillar deposits and of extracellular deposits ofthe β-amyloid peptide (A-β) forming amyloid plaques. (Yanker et al.Nature Med. Vol. 2 No. 8 (1996)). As well as these signs, there are manyother abnormal changes including a deterioration of the immune andinflammatory systems as well as a deterioration of mitochondrialfunction, which can lead to an increase in oxidative stress, activationof the mechanisms of apoptosis and ultimately to cell death.

Amyloid plaques are predominantly composed of A-β peptides with 40 or 42residues, which are generated during the proteolytic process of theβ-amyloid peptide precursor (APP) protein. The extracellular deposits ofA-β peptides represent the invariable early characteristic feature ofall forms of AD, including the familial forms (FAD). FADs appearrelatively early (between 40 and 60 years) and are due to mutations inthe gene of APP in 5% of cases of FAD (>20 families) with six single ordouble missense mutations; in the gene of presenilin 1 (PS 1) in 50 to70% of cases of FAD (>200 families) with more than 80 differentmutations identified to date; and in the gene of presenilin 2 (PS 2) infewer cases of FAD with 2 missense mutations described in 8 families.Mutations in these three genes have been shown to induce changes in theproteolysis of APP, which lead to overproduction of A-β and to the earlyappearance of the pathology and of symptoms that are similar to those ofthe sporadic forms of AD.

The neuronal toxicity of the amyloid plaques might reside in the highmolecular weight fibrils that are formed by aggregation of soluble A-βpeptides in fibrillar forms that are soluble initially (also calledprotofibrillar form) and are then converted to insoluble formsincorporated in the amyloid plaques. In fact, it was shown in vitro thatthe soluble A-β peptide aggregates progressively to a fibrillar form(i.e. which can be labelled with agents such as Congo Red or thioflavinS which recognize the beta-sheet tertiary structures of thepeptides/proteins), of high molecular weight (>200 kDa) but stillsoluble. Because this form is soluble, it is often called theprotofibrillar form, whereas the fibrils result from even greateraggregation, leading to loss of solubility. The protofibrillartransitional forms are generally regarded as the precursors of theamyloid fibres and might be responsible for the cellular dysfunction andthe neuronal loss in Alzheimer's disease and in other diseasesassociated with the aggregation of proteins.

It has been shown that the senile amyloid plaques (i.e. aggregated, alsocalled mature plaques) are correlated with the cognitive status ofAlzheimer's patients in contrast to the diffuse deposits of A-β peptidewhich are also widely present in unaffected patients. (Duyckaerts etal., Neurobiol. Aging 1997; 18: 33-42 and Jellinger et al., 1998;54:77-95). By targeting these senile amyloid plaques in particular, itis therefore possible to treat Alzheimer's disease more specifically andeffectively.

A great many treatments have been tried for preventing the formation ofthe A-β peptides, for example inhibitors of the proteolytic process ofAPP.

Immunotherapeutic strategies such as the administration of anti-A-βantibody (to reduce the amyloid deposits) or immunization with antigensof the A-β peptides (to promote a humoral response) have been tested inorder to reduce the size and density of the plaques.

For example, a method of treatment against Alzheimer's disease has beendescribed (U.S. Pat. No. 7,179,463), consisting of administering anantibody directed against a protofibril presenting an Arctic mutation inthe region coding for the A-β peptide.

No example of antibody has really been described. Moreover, nocomparison of the affinity of the antibodies for the peptides as afunction of the molecular weight of these peptides has been performed.Other patents (U.S. Pat. No. 6,761,888 and U.S. Pat. No. 6,750,324) havereferred to antibodies recognizing various epitopes along the amino acidsequence of the peptide A-β₄₂. An international application(WO2007/108756) has been filed concerning antibodies specific for theprotofibrils but the antibodies described recognize both the highmolecular weight A-β peptides and the medium-weight oligomers.Furthermore, there is no mention of the affinity of the antibodies forthe mature plaques relative to their affinity for the diffuse plaques.

Despite the current development of knowledge concerning Alzheimer'sdisease, there is still a need for compositions and methods of treatmentand/or prevention of this pathology limiting the secondary effects tothe maximum extent. Antibodies such as described in the presentapplication, humanized and specific for the protofibrillar form of theA-β peptides, aim to solve this problem. Permitting recognition of thesenile amyloid plaques but not the diffuse plaques, the antibodiesaccording to the invention recognize the pathological plaques much moreeffectively than antibodies recognizing all forms of Abeta, which willlargely be attached to the diffuse deposits or attached to the solubleforms of monomeric or low-molecular-weight A-β peptide.

Moreover, the fact that only the protofibrillar forms of the A-βpeptides are recognized and not the protofibrillar forms of otherproteins not linked to Alzheimer's disease avoids useless binding thatmay reduce the concentration of antibodies that are effective againstthe disease.

The murine antibody that has been humanized will be called antibody 13C3throughout the present application.

The sequences that can code for or constitute the humanized antibodiesaccording to the invention are shown in Table 2.

The present invention relates to a humanized antibody that bindsspecifically to the protofibrillar form of the A-β peptide, i.e. a highmolecular weight peptide.

In a more advantageous embodiment, the antibody binds to the A-β peptidehaving a molecular weight greater than 200, 300, 400 or 500 kDa.

According to one embodiment, the antibody according to the inventionbinds to the A-β peptides aggregated into senile plaques and not to thediffuse deposits of A-β peptides.

In an advantageous embodiment, the antibody according to the inventionbinds specifically to the protofibrillar form of the A-β peptide but notto the other proteins of amyloid structure (for example IAPP, IsletAmyloid Polypeptide).

The present invention also relates to a humanized antibody havingreduced effector functions, making it possible to limit adverse effectssuch as the development of microhaemorrhages and vasogenic oedemas.

In an advantageous embodiment, the antibody according to the inventionno longer possesses effector functions.

In an even more advantageous embodiment, the antibody is animmunoglobulin G 4 whose Fc domain has undergone mutations reducing theproduction of half-molecules.

In an even more advantageous embodiment, the antibody is animmunoglobulin G 4 whose Fc domain has undergone mutations reducing theeffector activity.

The present invention relates to a humanized antibody comprising atleast one CDR encoded by a nucleotide sequence having a sequenceidentical to one of the sequences SEQ ID NO: 9, 11, 13, 15, 17 and 19,or by sequences differing respectively by 1, 2, 3, 4 or 5 nucleotidesfrom these sequences.

The present invention also relates to a humanized antibody comprising atleast one CDR having a sequence identical to one of the sequences SEQ IDNO: 10, 12, 14, 16, 18 and 20.

In another embodiment, the antibody according to the invention comprisesat least one CDR whose sequence differs by one to two amino acidsrelative to one of the sequences SEQ ID NO: 10, 12, 14, 16, 18, 20 and32, inasmuch as the antibody maintains its binding specificity.

In an advantageous embodiment, the antibody comprises the CDRs encodedby the nucleotide sequences SEQ ID NO: 9, 11, 13, 15, 17 and 19, or bysequences differing respectively by 1, 2, 3, 4 or 5 nucleotides fromthese sequences.

In another advantageous embodiment, the antibody comprises the CDRs ofsequence SEQ ID NO: 10, 12, 14, 16, 18 and 20.

The antibody according to the invention can also comprise the CDRsencoded by the nucleotide sequences SEQ ID NO: 9, 11, 13, 31, 17 and 19or by sequences differing respectively by 1, 2, 3, 4 or 5 nucleotidesfrom these sequences.

In an advantageous embodiment, the antibody according to the inventioncomprises the CDRs of sequence SEQ ID NO: 10, 12, 14, 32, 18 and 20.

One object of the invention is the humanized antibody comprising theCDRs encoded by the nucleotide sequences SEQ ID NO: 9, 11, 29, 31, 17and 19 or by sequences differing respectively by 1, 2, 3, 4 or 5nucleotides from these sequences.

The invention also relates to a humanized antibody comprising the CDRsof sequence SEQ ID NO: 10, 12, 30, 32, 18 and 20.

In an advantageous embodiment, the antibody according to the inventioncomprises a variable part of its heavy chain (VH) encoded by a sequencehaving at least 80%, 85%, 90%, 95% or 99% identity with the sequence SEQID NO: 5 or the sequence SEQ ID NO 27.

In an advantageous embodiment, the antibody according to the inventioncomprises a variable part of its heavy chain (VH) comprising a sequencehaving at least 80%, 85%, 90%, 95% or 99% identity with the sequence SEQID NO: 6 or the sequence SEQ ID NO 28.

In an advantageous embodiment, the antibody according to the inventioncomprises a variable part of its light chain (VL) encoded by a sequencehaving at least 80%, 85%, 90%, 95% or 99% identity with the sequence SEQID NO: 7 or the sequence SEQ ID NO 23.

In an advantageous embodiment, the antibody according to the inventioncomprises a variable part of its light chain (VL) comprising a sequencehaving at least 80%, 85%, 90%, 95% or 99% identity with the sequence SEQID NO: 8 or the sequence SEQ ID NO 24.

In an even more advantageous embodiment, the antibody comprises a heavychain comprising a variable part (VH) encoded by one of the nucleotidesequences SEQ ID

NO 5 and SEQ ID NO 27.

In an even more advantageous embodiment, the antibody comprises a heavychain comprising a variable part (VH) of polypeptide sequence SEQ ID NO6 or SEQ ID NO 28.

In another embodiment, the antibody comprises a light chain comprising avariable part (VL) encoded by one of the nucleotide sequences SEQ ID NO7 and SEQ ID NO 23.

In another embodiment, the antibody comprises a light chain comprising avariable part (VL) of polypeptide sequence SEQ ID NO 8 or SEQ ID NO 24.

In an advantageous embodiment, the antibody comprises the sequencesencoded by the nucleotide sequences SEQ ID NO: 5 and 7.

In an advantageous embodiment, the antibody comprises the polypeptidesequences SEQ ID NO: 6 and 8.

In another embodiment, the antibody comprises the sequences encoded bythe nucleotide sequences SEQ ID NO: 5 and 23.

In another embodiment, the antibody comprises the polypeptide sequencesSEQ ID NO: 6 and 24.

In another embodiment, the antibody comprises the sequences encoded bythe nucleotide sequences SEQ ID NO: 27 and 23.

In another embodiment, the antibody comprises the polypeptide sequencesSEQ ID NO: 28 and 24.

The present invention also relates to an antibody comprising a heavychain encoded by a sequence having at least 80%, 85%, 90%, 95% or 99%identity with one of the nucleotide sequences SEQ ID NO 1 and SEQ ID NO25.

The present invention also relates to an antibody comprising a heavychain having at least 80%, 85%, 90%, 95% or 99% identity with thepolypeptide sequence SEQ ID NO 2 or with the polypeptide sequence SEQ IDNO 26.

In an advantageous embodiment the antibody comprises a light chainencoded by a sequence having at least 80%, 85%, 90%, 95% or 99% identitywith one of the nucleotide sequences SEQ ID NO 3 and SEQ ID NO 21.

In another embodiment the antibody comprises a light chain comprising asequence having at least 80%, 85%, 90%, 95% or 99% identity with one ofthe polypeptide sequences SEQ ID NO 4 and SEQ ID NO 22.

One object of the invention is an antibody comprising the sequencesencoded by the nucleotide sequences SEQ ID NO: 1 and 3.

Another object of the invention is an antibody whose sequence comprisesthe polypeptide sequences SEQ ID NO: 2 and 4.

One object of the invention is an antibody comprising the sequencesencoded by the nucleotide sequences SEQ ID NO: 1 and 21.

Another object of the invention is an antibody whose sequence comprisesthe polypeptide sequences SEQ ID NO: 2 and 22.

One object of the invention is an antibody comprising the sequencesencoded by the nucleotide sequences SEQ ID NO: 25 and 21.

Another object of the invention is an antibody whose sequence comprisesthe polypeptide sequences SEQ ID NO: 26 and 22.

Another object of the invention is a humanized anti-peptide Aβ antibodyhaving an affinity for the protofibrillar form of peptide Aβ at least100 times greater than its affinity for the other forms of this peptide.

Another object of the invention is an antibody, characterized in that itinduces a reduction of amyloid plaques.

Another object of the invention is the use of a humanized anti-peptideAβ antibody in the treatment of diseases associated withneurodegenerative disorders, and in particular in the treatment ofAlzheimer's disease.

Another object of the invention is a pharmaceutical compositioncomprising a humanized anti-peptide Aβ antibody and excipients.

Another object of the invention is a method of treatment of Alzheimer'sdisease comprising the administration of a humanized anti-peptide-Aβantibody to the patient.

Another object of the invention is a cell or cells producing a humanizedanti-peptide-Aβ antibody, as well as the method of production of thisantibody comprising the culturing of these cells. Said cells are derivedadvantageously from one cell line.

One object of the invention is a medicinal product comprising ahumanized anti-peptide-Aβ antibody.

One object of the invention is a polynucleotide coding for a polypeptidehaving at least 80%, 85%, 90%, 95% or 99% identity with one of thesequences SEQ ID NO: 2, 4, 6, 8, 22, 24, 26 or 28.

Another object of the invention is a polynucleotide with a sequencehaving at least 80%, 85%, 90%, 95% or 99% identity with one of thesequences SEQ ID NO: 1, 3, 5, 7, 21, 23, 25, or 27.

Another object of the invention is a recombinant vector comprising anucleic acid having one of the sequences SEQ ID NO 1, 3, 5, 7, 21, 23,25, or 27, as well as a host cell comprising this vector.

Definitions

Specific binding is understood as a difference by a factor of at leastabout 10, 20, 30, 40, 50, or 100 between the strength of binding to onereceptor relative to another, here between binding to the protofibrillarform of the A-β peptide and binding to the other forms of the peptide.

“Epitope” means the site of the antigen to which the antibody binds. Ifthe antigen is a polymer, such as a protein or a polysaccharide, theepitope can be formed by contiguous or non-contiguous residues. Here theepitope is conformational, i.e. related to the three-dimensionalstructure of the protofibrillar A-β peptide.

“Protofibrillar form” means an oligomeric form of A-β peptides, solublein vitro and which can be isolated as an entity of molecular weightgreater than 200 kDa, 300 kDa, 400 kDa or 500 kDa and which can fixagents such as thioflavin-S or Congo Red.

“Senile plaque” means a plaque composed of an amyloid core (fixingthioflavin S or Congo Red) surrounded by dystrophic neurites and areaction of glial cells. Senile plaques are found in particular inpatients with Alzheimer's disease, in contrast to the diffuse amyloiddeposits (which do not fix thioflavin S or Congo Red), which are farmore numerous but are not associated with the disease.

An antibody, also called immunoglobulin, is composed of two identicalheavy chains (“CH”) and two identical light chains (“CL”), which arejoined by a disulphide bridge.

Each chain contains a constant region and a variable region. Eachvariable region comprises three segments called “complementaritydetermining regions” (“CDRs”) or “hypervariable regions”, which aremainly responsible for binding to the epitope of an antigen.

The term “VH” refers to the variable regions of a heavy chain ofimmunoglobulin of an antibody, including the heavy chains of a fragmentFv, scFv, dsFv, Fab, Fab′ or F(ab)′.

The term “VL” refers to the variable regions of a light chain ofimmunoglobulin of an antibody, including the light chains of a fragmentFv, scFv, dsFv, Fab, Fab′ or F(ab)′.

“Antibody” also means any functional fragment of antibody: Fab (Fragmentantigen binding), Fv, scFv (single chain Fv), Fc (Fragment,crystallizable). Preferably, these functional fragments will befragments of type Fv, scFv, Fab, F(ab′) 2, Fab′, scFv-Fc, diabodies,multispecific antibodies (notably bispecific), synthetic polypeptidescontaining the sequences of one or more CDRs, which generally possessthe same specificity of fixation as the humanized antibody from whichthey are derived. According to the present invention, fragments ofantibodies of the invention can be obtained from the humanizedantibodies by methods such as digestion by enzymes, such as pepsin orpapain and/or by cleavage of the disulphide bridges by chemicalreduction.

Nanobodies also come under this definition.

“CDR or CDRs” denotes the hypervariable regions of the heavy and lightchains of the immunoglobulins as defined by Kabat et al. (Kabat et al.,Sequences of proteins of immunological interest, 5th Ed., U.S.Department of Health and Human Services, NIH, 1991, and later editions).There are 3 heavy-chain CDRs and 3 light-chain CDRs. The term CDR orCDRs is used here to denote, as applicable, one or more, or even all, ofthese regions that contain the majority of the amino acid residuesresponsible for the affine binding of the antibody for the antigen orthe epitope that it recognizes. The most conserved regions of thevariable domains are called FR regions or sequences, for “frameworkregions”.

The present invention relates to humanized antibodies.

“Humanized antibody” means an antibody that contains mainly humanimmunoglobulin sequences. This term generally refers to a non-humanimmunoglobulin that has been modified by incorporating human sequencesor residues found in human sequences.

In general, humanized antibodies comprise one or typically two variabledomains in which all or part of the CDR regions correspond to partsderived from the non-human parent sequence and in which all or part ofthe FR regions are derived from a human immunoglobulin sequence. Thehumanized antibody can then comprise at least one portion of a constantregion of immunoglobulin (Fc), in particular that of the selectedreference human immunoglobulin.

We thus try to obtain an antibody that is the least immunogenic in ahuman. Thus it is possible that one or two amino acids of one or moreCDRs are modified by an amino acid that is less immunogenic for thehuman host, without substantially reducing the binding specificity ofthe antibody to the A-β peptide of high molecular weight. Furthermore,the residues of the framework regions need not be human and it ispossible that they are not modified, as they do not contribute to theimmunogenic potential of the antibody.

Several methods of humanization are known by a person skilled in the artfor modifying a non-human parent antibody to an antibody that is lessimmunogenic in humans. Complete identity of the sequences with a humanantibody is not essential. In fact complete sequence identity is notnecessarily a predictive indicator of reduced immunogenicity andmodification of a limited number of residues can lead to humanizedantibodies presenting a very attenuated immunogenic potential in humans(Molecular Immunology (2007) 44, 1986-1998).

Some methods are for example the inclusion of CDRs (grafting) (EPO 0 239400; WO 91/09967; and U.S. Pat. Nos. 5,530,101 and 5,585,089), theresurfacing (EPO 0 592 106; EPO 0 519 596; Padlan, 1991, Molec Imm28(4/5):489-498; Studnicka et al., 1994, Prot Eng 7(6):805-814; andRoguska et al., 1994, PNAS 91:969-973) or chain mixing (U.S. Pat. No.5,565,332).

The present invention relates in particular to humanized antibodieswhose variable parts are modified according to the technology explainedin international patent application WO 2009/032661.

This technique notably uses dynamic molecular simulation based onthree-dimensional models of antibodies, said models being constructed byhomology.

The present invention also relates to any form of antibody havingdiminished effector functions, such as immunoglobulins bearing mutationsof the Fc domain reducing its affinity for the receptors of the immunesystem or such as nanobodies.

“Effector functions” means any fixation of the Fc domain of the antibodyto receptors or proteins inducing immune responses. Decreasing theseeffector functions makes it possible to reduce adverse effects such asthe induction of microhaemorrhages (Racke et al. J Neurosci 2005,25:629).

Affinity can be measured by any technique known by a person skilled inthe art. It is advantageously measured by the Biostat Speed techniquedeveloped on the basis of the algorithms described by Ratkovsk D A andReedy T J (Biometrics, 1986, 42, 575-82).

In order to permit expression of heavy chains and/or light chains of theantibody according to the invention, the polynucleotides coding for saidchains are inserted in expression vectors. These expression vectors canbe plasmids, YACs, cosmids, retroviruses, episomes derived from EBV, andall the vectors that a person skilled in the art may judge to besuitable for expression of said chains.

These vectors can be used for transforming cells advantageously derivedfrom one cell line. Said cell line is even more advantageously derivedfrom a mammal. It is advantageously the CHO line or a line derived fromthis line, or the HEK293 line or a line derived from this line.

The transformation of the cells can be carried out by any method knownby a person skilled in the art for introducing polynucleotides into ahost cell. Said method can be transformation by means of dextran,precipitation by calcium phosphate, transfection by means of polybrene,protoplast fusion, electroporation, encapsulation of the polynucleotidesin liposomes, biolistic injection and direct micro-injection of DNA intothe nucleus.

The antibody according to the invention can be included inpharmaceutical compositions with a view to administration by thetopical, oral, parenteral, intranasal, intravenous, intramuscular,subcutaneous, intraocular or other routes. Preferably, thepharmaceutical compositions contain pharmaceutically acceptable vehiclesfor an injectable formulation. These can be in particular sterile,isotonic saline solutions (monosodium or disodium phosphate, sodium,potassium, calcium or magnesium chloride, etc., or mixtures of saidsalts), or dry compositions, notably lyophilized, which, by addingsterilized water or physiological serum as appropriate, permitinjectable solutes to be constituted.

As an example, a pharmaceutical composition comprises (1) a Dulbeccophosphate buffer (pH-7.4), optionally containing 1 mg/ml to 25 mg/ml ofhuman serum albumin, (2) 0.9% w/v of sodium chloride (NaCl), and (3) 5%(w/v) of dextrose. It can also comprise an antioxidant such astryptamine and a stabilizer such as Tween 20.

The pathologies in question can be any diseases associated with thedeposition of amyloid plaques. In particular, the pathology in questionis Alzheimer's disease.

The doses depend on the desired effect, the duration of the treatmentand the route of administration used; they are generally between 5 mgand 1000 mg of antibody per day for an adult. Generally the doctor willdetermine the appropriate dosage in relation to the stage of thedisease, the patient's age and weight, or any other patient-relatedfactor that has to be taken into account.

The present invention is illustrated, but is not limited, by theexamples given below.

BRIEF DESCRIPTION OF THE DRAWINGS:

FIG. 1A: Map of the plasmid pXL4973 permitting expression of the lightchain LC1 of the antiAbeta antibody 13C3-VH1VL1.

FIG. 1B: Map of the plasmid pXL4979 permitting expression of the heavychain HC1 of the antiAbeta antibody 13C3-VH1VL1.

FIGS. 2A and 2B: Separation of the protofibrils and of thelow-molecular-weight oligomers by gel filtration on Superdex 75 (at t=0and at t=16 h respectively).

FIG. 3: Determination of the molecular weight of the protofibrils.

FIGS. 4A, 4B and 4C: Determination of the affinities of the humanizedantibodies (antibodies LP09027 (4A), LP09026 (4B) and LP09028 (4C)respectively) for the protofibrils (mean value from 3 experiments±sem).

FIG. 5: Specificity of the humanized antibody LP09027 with respect tofibrils of Aβ.

FIGS. 6A and 6B: Specificity of the humanized antibody (LP09027) for themature senile plaques respectively of the frontal cortex (6A) and of thehippocampus (6B) of a mouse. The arrows indicate the senile plaques.

EXAMPLES Example 1 Obtaining Humanized Antibodies

A murine antibody 13C3 was humanized.

This example describes the sequence and the production of the humanizedanti-peptide Aβ antibody VH1VL1 (LP09027) by production by transientexpression in the mammalian line HEK293 designated FreeStyle 293-F.

The cDNAs coding for the humanized variable chains VL1 and VH1 are fusedwith the cDNAs coding for the human constant regions Ckappa and IgG4respectively. The sequence of the constant region IgG4 is that of thevariant having the substitutions S241 P and L248E in Kabat'snomenclature, for a significant reduction in the production ofhalf-molecules (Angla et al., 1993, Mol. Immunol., 30: 105-108) and theeffector functions (WO 97/09351).

The nucleic acid sequences coding for CH1 (SEQ ID NO 1) and for CL1 (SEQID NO 3) were cloned independently in the expression vector to generatethe plasmids pXL4973 (FIG. 1A) and pXL4979 (FIG. 1B), respectively.

A batch of the antibody is produced by production by transientexpression in the line FreeStyle 293-F (Invitrogen) afterco-transfection of the plasmids pXL4973 and pXL4979 according to theprotocol described by Invitrogen (catalogue reference K9000-01). Thisbatch (LP09027) is then purified by affinity chromatography on a columnof MabSelect gel (Amersham) according to the supplier's recommendationsand then formulated in PBS buffer (reference Dulbecco 14190-094) andsubmitted to sterile filtration (0.2 μm). Starting from 1 L of culture,33 mg of antibody is obtained at a purity of 97% by SDS-PAGE indenaturing conditions and by steric exclusion chromatography. The massobtained by SDS-PAGE in denaturing conditions and by LC/MS is inagreement with the primary amino acid sequence and the presence of anN-glycan on the Fc domain, namely a mass of 23969 Da for LC1 and 49650Da for HC1 taking into account the N-glycan in the G0F form. The massobtained by SDS-PAGE in non-denaturing conditions and by size exclusionchromatography is in agreement with the hetero-tetrameric structure ofthe antibody of 150 kDa (FIG. 4A).

According to the same method, batches of humanized antibodies LP09026and LP09028 were produced starting from the nucleotide sequences SEQ IDNO 25 and SEQ ID NO 21 for LP09026 (FIG. 4B), and SEQ ID NO 1 and SEQ IDNO 21 for LP09028 (FIG. 4C).

Example 2 Preparation of Protofibrils from Peptide Aβ (1-42)

The protofibrils were prepared from the synthetic peptide Aβ (1-42)according to the method described by Johansson et al. (FEBS, 2006,2618-2630). The lyophilized peptide (Anaspec reference 24224) isdissolved in 10 mM NaOH at a concentration of 100 μM, then stirred for 1min and incubated on ice for 10 min. The solution of peptide is thendiluted in buffer of 100 mM sodium phosphate, 200 mM NaCl pH=7.4 to aconcentration of 50 μM, then stirred for 1 min. The preparation isincubated overnight at 37° C. for formation of protofibrils and thencentrifuged at 17900 g for 15 min at 16° C. to remove the insolubleaggregates. To separate the protofibrils from the oligomeric forms of Aβof low molecular weight, the supernatant is loaded on a Superdex 75 gelfiltration column equilibrated in 50 mM ammonium acetate buffer pH=8.5.The fractions corresponding to the protofibrils and to thelow-molecular-weight oligomers are collected and stored at 4° C. FIG. 2shows a typical profile of separation of the protofibrils. The molecularweight of the protofibrils is determined by Superdex200 gel filtrationusing, as markers of molecular weight, the Biorad calibration kit(reference 150-1901). FIG. 3 shows that the molecular weight of theprotofibrils is greater than 200 kDa.

Example 3 Specificity and Affinity of the Humanized Antibodies withRespect to the Protofibrils

50 μl of protofibrils and low-molecular-weight oligomers at aconcentration of 1 μg/ml in PBS (Gibco, reference 70011) are depositedin the wells of an ELISA plate (Nunc, reference 442404) and incubatedovernight at 4° C. After removing the excess antigen, 200 μl of bufferPBS+5% milk powder (weight/volume) is deposited in each well to removethe non-specific adsorptions and incubated for 2 h at room temperature.The wells are then washed 4 times with 300 μl of buffer PBS Tween 0.02%.50 μl of a primary antibody solution (dilution of 3 in 3 in PBS Tweenstarting from a concentration of 100 μg/ml for the oligomers and from 25μg/ml for the protofibrils) is added to each well and incubated for 1 hat room temperature. The wells are washed 4 times with 300 μl of bufferPBS Tween. The secondary anti-Fc human antibody coupled to peroxidase(Goat Anti Human IgG (Fc) peroxidase conjugated, Pierce, reference31413) diluted to 1/10000 in buffer PBS Tween is added to each well andincubated for 1 h at room temperature. After 4 washings with 300 μl ofPBS Tween, 100 μl of TMB (Interchim, reference UP664782) is added toeach well and incubated for about 10 min, then the reaction is stoppedwith a solution of 1M HCl (Interchim, reference UPS29590) and the platesare read at an OD measured at a wavelength of 450 nm. The EC50 valuesare determined by BioStat Speed. The results obtained are presented inTable 1 and in FIG. 4 and show the very high specificity of the antibodyfor the protofibrils relative to the low-molecular-weight oligomers(factor of 184).

TABLE 1 EC50 (μg/ml) LMW PF LMW/PF LP09026  41.4 ± 40.1 0.0587 ± 0.004705.3 LP09027 14.7 ± 2.7 0.0798 ± 0.007 184.2 LP09028 21.8 ± 5.3 0.0892± 0.007 244.4

The lyophilized peptide Aβ1-42 (Anaspec reference 24224) is dissolvedaccording to the supplier's recommendations: 40 μl of 1% NH4OH is addedto 500 μg of Aβ1-42.

After complete dissolution, 460 μl of PBS is added to obtain aconcentration of 1 mg/ml. Aliquots of 10 μl are prepared and stored at−80° C.

50 μl of a solution of peptide Aβ1-42 at a concentration of 1 μg/ml incarbonate buffer (NaHCO₃ 0.025 M (Acros Organics, reference 217120010),Na₂CO₃ 0.025 M (Acros Organics, reference 207810010), pH 9.7 isdeposited in the wells of an ELISA plate and incubated overnight at roomtemperature. As previously, the wells are washed with buffer PBS Tween,incubated in the presence of buffer PBS+5% milk powder (weight/volume)and washed with buffer PBS Tween. The humanized antibody at aconcentration of 0.02 μg/ml is incubated for 1 h at room temperaturewith a concentration range (starting from 1 μg/ml) of peptides Aβ1-28(Bachem, reference H7865), Aβ1-16 (Anaspec, reference 24225), Aβ25-35(Anaspec, reference 24227), low-molecular-weight oligomers orprotofibrils prepared as described previously. The antibody/antigenmixture is then deposited in each well and the microtitration plate isincubated for 1 h at room temperature. The free, uncomplexed antibody isdetermined according to the same ELISA protocol as described previously.These competitive experiments show that only the protofibrils with amuch higher affinity than the low-molecular-weight oligomers are capableof neutralizing the humanized antibody by preventing it from interactingwith the peptide Aβ1-42; none of the peptides is capable of neutralizingthe antibody.

Example 4 Specificity of the Humanized Antibody LP09027 with Respect tothe Fibrils of Aβ1-42

The peptide Aβ1-42 (Anaspec, 20276) is dissolved in 200 μl of 10 mM NaOHto a concentration of 5 mg/ml. The peptide IAPP (Anaspec, 60804) isdiluted in 200 μl of 50% DMSO to a concentration of 5 mg/ml. 100 μl ofeach preparation is diluted in 400 μl of PBS 1.25×. The finalconcentration of the peptides is 1 mg/ml in 500 μl. The samples areincubated for 72 h at 37° C. After incubation, the samples arecentrifuged at 17900 g for 30 minutes at 4° C. The supernatant isremoved and the pellet is washed 3 times with PBS 1×. After the lastwashing, the pellet of fibrils is taken up in 150 μl of PBS. To checkfor the presence of fibrils of amyloid type, a thioflavin T fluorescencetest (Anaspec, 88306) is carried out. 20 μl of thioflavin T (20 μMfinal), 10 μl of the sample and 70 μl of PBS 1× (final volume 100 μl)are mixed in a well of a black plate (Corning, 3792). The thioflavin Tis excited at 450 nm and, in the presence of a structure of amyloidtype, emits fluorescence at 482 nm. 50 μl of fibrils of Aβ1-42 at 1μg/ml and IAPP at 0.5 μg/ml are deposited in each well of amicrotitration plate. The ELISA protocol is applied using serialdilutions of the humanized antibody starting from 10 μg/ml. FIG. 5 showsthat the humanized antibody LP09027 specifically recognizes the fibrilsof Aβ1-42 but not those of IAPP.

Example 5 Specificity of the Humanized Antibody LP09027 for the MatureSenile Plaques but not for the Diffuse Plaques

The humanized antibody (LP09027) conjugated with digoxigenin(digoxigenin-3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxysuccinimideester: Roche 11333054001; 11418165001) was used in immunohistochemistry(Ventana Robot) on brain sections from mice APP PS1 (Alzheimer modeldescribed by Schmitz C. et al., Am. J. Pathol, 2004, 164, 1495-1502) aswell as human brain sections (cerebral cortex) derived from patientswith Alzheimer's disease. The samples had been fixed in formol andembedded in paraffin beforehand.

The results obtained in the mouse (FIGS. 6A and 6B) clearly show thatthe humanized antibody recognizes exclusively the dense, mature senileplaques, but not the diffuse deposits of peptide Aβ.

These data correlate with the properties of this antibody, which isspecific for the protofibrillar Abeta form and so does not recognize thesoluble, mono- or oligomeric forms of this peptide.

TABLE 2 Nucleotide sequences Protein sequences Antibody 1 VH1VL1 VH₁ +CH₁ SEQ ID NO 1 SEQ ID NO 2 VL₁ + CL₁ SEQ ID NO 3 SEQ ID NO 4 VH₁ SEQ IDNO 5 SEQ ID NO 6 VL₁ SEQ ID NO 7 SEQ ID NO 8 CDR VH₁ SEQ ID NO 9, 11, 13SEQ ID NO 10, 12, 14 CDR VL₁ SEQ ID NO 15, 17, 19 SEQ ID NO 16, 18, 20Antibody 2 VH1 VL2 VH₁ + CH₁ SEQ ID NO 1 SEQ ID NO 2 VL₂ + CL₂ SEQ ID NO21 SEQ ID NO 22 VH₁ SEQ ID NO 5 SEQ ID NO 6 VL₂ SEQ ID NO 23 SEQ ID NO24 CDR VH₁ SEQ ID NO 9, 11, 13 SEQ ID NO 10, 12, 14 CDR VL₂ SEQ ID NO31, 17, 19 SEQ ID NO 32, 18, 20 Antibody 3 VH2 VL2 VH₂ + CH₂ SEQ ID NO25 SEQ ID NO 26 VL₂ + CL₂ SEQ ID NO 21 SEQ ID NO 22 VH₂ SEQ ID NO 27 SEQID NO 28 VL2 SEQ ID NO 23 SEQ ID NO 24 CDR VH₂ SEQ ID NO 9, 11, 29 SEQID NO 10, 12, 30 CDR VL₂ SEQ ID NO 31, 17, 19 SEQ ID NO 32, 18, 20

1.-37. (canceled)
 38. A cell producing a humanized antibody specific forthe protofibrillar form of the A-β peptide, wherein the antibodycomprises at least one CDR having a sequence identical to one of thesequences set forth as SEQ ID NO: 10, 12, 14, 16, 18, 20, 30, or
 32. 39.Method of producing a humanized antibody specific for the protofibrillarform of the A-β peptide, the method comprising culturing the cell ofclaim 38 to produce the antibody.
 40. (canceled)
 41. A polynucleotideencoding a polypeptide having at least 80% identity with a sequenceselected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 22, 24, 26and
 28. 42. A polynucleotide comprising at least 80% identity with asequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7,21, 23, 25, and
 27. 43. A recombinant vector comprising thepolynucleotide of claim
 41. 44. A host cell comprising the vector ofclaim
 43. 45. The cell according to claim 38, wherein the antibodyspecific for the protofibrillar form of the A-β peptide comprises sixCDRs set forth as SEQ ID NOs: 10, 12, 14, 32, 18, and
 20. 46. The cellaccording to claim 45, wherein the antibody specific for theprotofibrillar form of the A-β peptide comprises a heavy chain variableregion set forth as SEQ ID NO: 6 and a light chain variable region setforth as SEQ ID NO:
 24. 47. The cell according to claim 45, wherein theantibody specific for the protofibrillar form of the A-β peptidecomprises a heavy chain variable region comprising a sequence having atleast 95% identity with SEQ ID NO: 6 and a light chain variable regioncomprising a sequence having at least 95% identity with SEQ ID NO: 24.48. The cell according to claim 45, wherein the antibody specific forthe protofibrillar form of the A-β peptide comprises a variable regionof a heavy chain comprising a sequence having at least 99% identity withSEQ ID NO: 6 and a variable region of a light chain comprising asequence having at least 99% identity with SEQ ID NO:
 24. 49. The cellaccording to claim 45, wherein the antibody specific for theprotofibrillar form of the A-β peptide comprises a heavy chain set forthas SEQ ID NO: 2 and a light chain set forth as SEQ ID NO:
 4. 50. Thecell according to claim 38, wherein the antibody specific for theprotofibrillar form of the A-β peptide comprises six CDRs set forth asSEQ ID NO: 10, 12, 30, 32, 18, and
 20. 51. The cell according to claim50, wherein the antibody specific for the protofibrillar form of the A-βpeptide comprises a heavy chain variable region set forth as SEQ ID NO:28 and a light chain variable region set forth as SEQ ID NO:
 24. 52. Thecell according to claim 50, wherein the antibody specific for theprotofibrillar form of the A-β peptide comprises a heavy chain variableregion comprising a sequence having at least 95% identity with SEQ IDNO: 28 and a light chain variable region comprising a sequence having atleast 95% identity with SEQ ID NO:
 24. 53. The cell according to claim50, wherein the antibody specific for the protofibrillar form of the A-βpeptide comprises a variable region of a heavy chain comprising asequence having at least 99% identity with SEQ ID NO: 28 and a variableregion of a light chain comprising a sequence having at least 99%identity with SEQ ID NO:
 24. 54. The cell according to claim 50, whereinthe antibody specific for the protofibrillar form of the A-β peptidecomprises a heavy chain set forth as SEQ ID NO: 26 and a light chain setforth as SEQ ID NO:
 22. 55. The cell according to claim 38, wherein theantibody specific for the protofibrillar form of the A-β peptidecomprises six CDRs set forth as SEQ ID NO: 10, 12, 14, 16, 18, and 20.56. The cell according to claim 55, wherein the antibody specific forthe protofibrillar form of the A-β peptide comprises a heavy chainvariable region set forth as SEQ ID NO: 6 and a light chain variableregion set forth as SEQ ID NO:
 8. 57. The cell according to claim 55,wherein the antibody specific for the protofibrillar form of the A-βpeptide comprises a heavy chain variable region comprising a sequencehaving at least 95% identity with SEQ ID NO: 6 and a light chainvariable region comprising a sequence having at least 95% identity withSEQ ID NO:
 6. 58. The cell according to claim 55, wherein the antibodyspecific for the protofibrillar form of the A-β peptide comprises avariable region of a heavy chain comprising a sequence having at least99% identity with SEQ ID NO: 6 and a variable region of a light chaincomprising a sequence having at least 99% identity with SEQ ID NO: 24.59. The cell according to claim 55, wherein the antibody specific forthe protofibrillar form of the A-β peptide comprises a heavy chain setforth as SEQ ID NO: 2 and a light chain set forth as SEQ ID NO: 4.